Protocol for analyzing transforming growth factor β signaling in dextran-sulfate-sodium-induced colitic mice using flow cytometry and western blotting

Summary Transforming growth factor β (TGF-β) is critical to the maintenance of intestinal immune homeostasis. Here, we present techniques for analyzing Smad molecules downstream of TGF-β receptor signaling in dextran-sulfate-sodium-induced colitic mice. We describe colitis induction, cell isolation, and flow cytometric cell sorting of dendritic cells and T cells. We then detail intracellular staining of phosphorylated Smad2/3 and western blotting analysis of Smad7. This protocol can be performed on a limited number of cells from many sources. For complete details on the use and execution of this protocol, please refer to Garo et al.1


SUMMARY
Transforming growth factor b (TGF-b) is critical to the maintenance of intestinal immune homeostasis. Here, we present techniques for analyzing Smad molecules downstream of TGF-b receptor signaling in dextran-sulfate-sodium-induced colitic mice. We describe colitis induction, cell isolation, and flow cytometric cell sorting of dendritic cells and T cells. We then detail intracellular staining of phosphorylated Smad2/3 and western blotting analysis of Smad7. This protocol can be performed on a limited number of cells from many sources. For complete details on the use and execution of this protocol, please refer to Garo et al. 1

BEFORE YOU BEGIN
Dysregulation of TGF-b signaling is associated with several inflammatory disorders including intestinal inflammation in animal models and inflammatory bowel diseases (IBD) in humans. 2,3 The amplitude of TGF-b signaling is tightly regulated by Smad proteins. 2,3 While Smad7 limits TGF-b signaling, Smad2 and Smad3 promote TGF-b signaling and prevent tissue inflammation and IBD. 2,3 Below, we describe a detailed protocol for the analysis of Smad molecules. The phospho flow cytometry approach we describe can be applied to detect a variety of phospho proteins in other signaling pathways at the cellular level with even a small number of cells as compared to timeconsuming traditional approaches requiring higher cell numbers. 2,4 Further, these methods can also be applied to a variety of cell lines and primary cells such as peripheral blood mononuclear cells (PBMCs).
2. Label 100 3 15 mm style petri dishes, sterile 50 mL centrifuge tubes (five tubes per mouse), sterile 25cm 2 vented cap, TC treated tissue culture flasks (two per mouse), and 2 mL Eppendorf tubes (one per mouse). 3. Place 100 mm cell strainer on one 50 mL conical tube per mouse and a 40 mm cell strainer on one 50 mL conical tube per mouse.
On the day of the experiment, before euthanization. 4. Set the incubator shaker at 37 C. 5. Add 0.077 g DTT to Buffer C and warm up 10 mL of Buffer C per mouse at 37 C. 6. Warm up 10 mL per mouse of the X-VIVO 15 Serum-Free medium with Gentamicin and Phenol Red at 37 C. 7. Fill two sets of 50 mL conical tubes with Buffer A and keep them on ice.
CRITICAL: Animal work can only be performed after appropriate approval is obtained and under the applicable guidelines and regulations.

Institutional permissions
All experiments were performed by guidelines from the Institutional Animal Care and Use Committee at Brigham and Women's Hospital.

Induction and scoring of DSS-induced colitis in mice
Timing: 8 days 8. Add 7.5 g of DSS to 250 mL of autoclaved drinking water for a final concentration of 3% (w/v).
a. Give control mice autoclaved drinking water without DSS. b. Do not allow animals access to any other water source during the seven days.
9. Weigh mice daily. a. Weight loss greater than 25% of initial weight is considered an endpoint. b. Score each mouse daily according to the following criteria during the DSS treatment period to evaluate the clinical severity of colitis.
Note: The disease activity index (DAI) is the combined score of three parameters: weight loss compared to initial weight, the presence of blood in stool, and stool consistency.
i. Weight Loss.

Score
Level of weight loss

MATERIALS AND EQUIPMENT
Deoxyribonuclease I (DNase I): Reconstitute 100mg in 50 mL sterile H 2 O (2 mg/mL). Store in 1 mL aliquots at À20 C for up to six months. Liberase TL: Reconstitute 5 mg with 2 mL 13 PBS (2.5 mg/mL). Store in 1 mL aliquots at À20 C for up to six months. Buffer A: 500 mL HBSS; 25 mL FCS; 12.5 mL 1M HEPES (store at 4 C for up to 6 months). Buffer B: 500 mL HBSS; 2 mL 0.5M EDTA; 12.5 mL 1M HEPES (store at 4 C for up to 6 months). FACS Buffer: 1000 mL (13) PBS with 20 mL (2%) of fetal calf serum (store at 4 C for up to 6 months). Enzyme solution: 1.25 mL of liberase TL (2.5 mg/mL) and 1 mL DNase I (2 mg/mL) to 100 mL of prewarmed X-VIVO 15 media (Store at 37 C and use the same day). This step describes dissection of the colon from colitic mice and the isolation of cells from the lamina propria, a layer of connective tissue that houses many of the gut's immune cells. This layer will be digested, and the cells isolated using enzymatic digestion.
1. Dissect the entire length of the colon by making an incision at the anus and another immediately below the cecum. Remove the colon and wash it by briefly submerging it in 13 PBS to remove any excess blood. 2. Lay the colon flat on a paper towel and remove fecal contents by pushing them out using forceps. a. Wet the forceps to make it easier to handle the colon and squeeze out the fecal contents. b. Clean the forceps after every sample. 3. Remove as much fat attached to the colon as possible with forceps. 4. Cut the colon vertically and fold open the tissue to expose the lumen. 5. Transfer the colon into a 50 mL conical tube containing ice-cold Buffer A. 6. Vortex the tube for 10 s and then store it on ice. 7. Once all colons are collected and stored in the 50 mL conical tube containing Buffer A, add 10 mL of the pre-warmed Buffer C containing DTT to a 25 cm 2 flask (one per tissue). 8. Place a metal tea strainer on a large beaker. Briefly, vortex each 50 mL tube with colon tissue in Buffer A and then pour it out over the strainer to remove the buffer. 9. Lay the colon tissue flat on a paper towel for a few seconds to remove any remaining buffer. 10. Transfer the colon tissue to the flask containing 7.5 mL of the pre-warmed Buffer C with DTT.
CRITICAL: For multiple samples, work quickly to minimize the time difference between samples and to avoid longer digestions for earlier samples.
11. Firmly close the flasks now containing the tissue with caps and cover them tightly with parafilm to prevent leakage. 12. Horizontally place the sealed flasks with the colons into a 37 C incubating shaker immediately and secure them using tape to prevent them from falling. 13. Incubate for 20 min in the incubator at a shaking speed of 180 rpm.
a. If the incubator isn't at 37 C prior to adding samples, add 5 extra minutes to each incubation. 14. During the incubation, thaw the Liberase and DNase I aliquots at 37 C. Once thawed, prepare 10 mL per mouse of the enzyme solution (scale up as needed). Keep the enzyme solution at 37 C. 15. When the incubation is over, place the flasks on ice immediately to stop the reaction. 16. Lay the colon tissue on a paper towel for a few seconds to remove buffer. 17. Transfer the colon tissue to another 50 mL conical tube containing 10 mL of ice-cold Buffer A. 18. Vortex briefly, then pour buffer and colon over the tea strainer.  Note: Smad proteins can also be detected in DCs and CD4+ T cells isolated from other organs such as the mesenteric lymph nodes (MLN) and the spleen from colitic and non-colitic naive mice. If using bone marrow-derived DCs or ex vivo-isolated DCs from spleen or LN and CD4+ T cells from naïve mice, stimulate these cells with TGF-b for at least 30 min to 1 h. CD4+ T cells should be cultured with anti-CD3 and anti-CD28 antibodies for at least 12 h before adding TGF-b. Please note that as of this writing, no commercially available flow antibody is available for Smad7. Troubleshooting.

Intracellular staining for phosphorylated Smad2/3
Timing: 3 h Note: Keep cells on ice and protected from light until sorting begins. Troubleshooting.

Timing: 2 days
This step describes how western blotting was used to analyze Smad7 protein expression in FACSorted DCs and T cells from colonic lamina propria.
63. Following initial FACS sorting from the Lamina Propria or following subsequent treatment with TGF-b in culture, collect cells in a tube and centrifuge at 1,000 3 g for 5 min at 4 C.
CRITICAL: Protein degradation is maximum at room temperature. Thus, samples should be kept on ice during processing and at À80 C for storage.
Pause point: The cell pellet can be stored at À80 C for several weeks.
64. Wash the pellet with PBS. Note: This will be the whole cell lysate.
Note: The RIPA buffer should be used for both the blank and the standard for protein estimation. Pause point: The total protein can be stored at À80 C for several weeks.
71. Take 30-50 mg total protein and add 6 3 loading buffer. Make up the volume to 10 mL using lysis buffer. 72. Heat the samples at 100 C for 10 min and keep them on ice immediately. 73. Centrifuge at 7,000 3 g for 10 min at RT. 74. Load 10 mL of the protein sample per lane on a gradient of 4%-20% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gel. 75. Load 10 mL for pre-stained protein standard as a marker.Run a the SDS-PAGE gel at 100 V until the markers are separated. 76. Immerse a PVDF membrane for 20 s in methanol during the SDS-PAGE and incubate in the transfer buffer. 77. Immerse blotting papers in the transfer buffer for 10 min or more. 78. Take out the gel and immerse it in the transfer buffer. 79. Rock gently for 5-10 min. 80. Place the PVDF membrane gently on the gel. Then, place blotting papers on both sides of the PVDF membrane and the gel. Carefully remove bubbles using a roller. 81. Using the wet transfer apparatus, transfer proteins onto the PVDF membrane at 300 mA for 60 min. 82. Wash PVDF membrane twice in Tris buffered saline with 0.1% Tween 20 (TBST) after the transfer. 83. Use 5% non-fat milk in TBST for blocking at room temperature for 60 min on a rocker. 84. Incubate with Smad7 antibody (1:500 dilution) or GAPDH (1:5000 dilution) or a-tubulin (1:5000 dilution) in 5% non-fat milk in TBST overnight at 4 C. 85. After incubation with the primary antibody, rinse the PVDF membrane twice with PBST. Then, wash the membrane thrice with TBST at room temperature for 10 min on a rocker. 86. Incubate the PVDF membrane with the secondary antibody conjugated with HRP in 5% non-fat milk in TBST (1: 5,000 dilution) at room temperature for 1 h on a rocker. 87. Rinse the PVDF membrane twice with TBST. Then, wash the PVDF membrane thrice by TBST at room temperature for 10 min each. 88. Decant the TBST and add the Luminata HRP reagent. Incubate for 5 min. 89. Image on the G:Box ChemiXX6/XX9 -High-resolution gel imaging system. 90. Export images in 300 dpi tiff format for creating the figures. See Figure 3 for representative western data.
Note: Please note that as of this writing, no commercially available flow antibody is available for Smad7. Troubleshooting.

EXPECTED OUTCOMES
This method provides step-by-step instructions for the analysis of SMAD molecules in the colonic lamina propria of colitic mice. Smad molecules are expected to be increased in both DCs and CD4+ T cells during DSS-induced colitis (Figures 2 and 3).

LIMITATIONS
One limitation of the current protocol is that it has only been optimized for these particular SMAD molecules. Analysis of other SMAD molecules or phopho proteins in a different signaling pathway may require optimization. Additionally, it is imperative that the isolation of the colonic lamina propria cells and subsequent flow staining is performed as quickly as possible to maximize viability. Smad proteins can also be detected in DCs and CD4 + T cells isolated from other organs such as the mesenteric lymph nodes (MLN) and the spleen from colitic and non-colitic naive mice. If using bone marrow-derived DCs or ex vivo-isolated DCs from spleen or LN and CD4 + T cells from naïve mice, stimulate these cells with TGF-b for at least 30 min to 1 h. CD4+ T cells should be cultured with anti-CD3 and anti-CD28 antibodies for at least 12 h before adding TGF-b.

OPEN ACCESS
TROUBLESHOOTING Problem 1 Low cell viability after flow staining (related to step 48).

Potential solution
Work quickly when processing multiple samples to avoid longer digestions for earlier samples. Keep samples on ice to maximize viability.

Problem 2
Low expression of Smad molecules by flow cytometry (related to step 62).

Potential solution
Smad molecules are difficult to detect by flow cytometry. Therefore, it is ideal to reserve the brightest available fluorophores for these markers.

Problem 3
Degradation of Smad molecules (related to step 63).

Potential solution
Protein degradation is maximum at room temperature. Thus, samples should be kept on ice during processing and at À80 C for storage.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Gopal Murugaiyan (mgopal@rics.bwh.harvard.edu).

Materials availability
This study did not generate any unique reagents. Western blot of Smad7 in CD11c + DCs and CD4 + T cells isolated ex vivo from mesenteric lymph nodes (MLN) of naïve and colitic WT mice after 7 days 3% DSS in drinking water. Western blots were quantitated using the quantitation software Genetools from Syngene. Briefly, the intensity of the blots was measured by adding a box of equal area to cover individual blots, and the equal box was used for the background subtraction. Smad7 intensities were normalized to its respective Gapdh, and fold change was calculated with respect to naïve DCs. Data represent mean G SD. * represents p < 0.05. Adapted from reference. Data and code availability This study did not generate datasets/codes.